Development and characterization of a Gucy2d-cre mouse to selectively manipulate a subset of inhibitory spinal dorsal horn interneurons

Recent transcriptomic studies identified Gucy2d (encoding guanylate cyclase D) as a highly enriched gene within inhibitory dynorphin interneurons in the mouse spinal dorsal horn. To facilitate investigations into the role of the Gucy2d+ population in somatosensation, Gucy2d-cre transgenic mice were created to permit chemogenetic or optogenetic manipulation of this subset of spinal neurons. Gucy2d-cre mice created via CRISPR/Cas9 genomic knock-in were bred to mice expressing a cre-dependent reporter (either tdTomato or Sun1.GFP fusion protein), and the resulting offspring were characterized. Surprisingly, a much wider population of spinal neurons was labeled by cre-dependent reporter expression than previous mRNA-based studies would suggest. Although the cre-dependent reporter expression faithfully labeled ~75% of cells expressing Gucy2d mRNA in the adult dorsal horn, it also labeled a substantial number of additional inhibitory neurons in which no Gucy2d or Pdyn mRNA was detected. Moreover, cre-dependent reporter was also expressed in various regions of the brain, including the spinal trigeminal nucleus, cerebellum, thalamus, somatosensory cortex, and anterior cingulate cortex. Injection of AAV-CAG-FLEX-tdTomato viral vector into adult Gucy2d-cre mice produced a similar pattern of cre-dependent reporter expression in the spinal cord and brain, which excludes the possibility that the unexpected reporter-labeling of cells in the deep dorsal horn and brain was due to transient Gucy2d expression during early stages of development. Collectively, these results suggest that Gucy2d is expressed in a wider population of cells than previously thought, albeit at levels low enough to avoid detection with commonly used mRNA-based assays. Therefore, it is unlikely that these Gucy2d-cre mice will permit selective manipulation of inhibitory signaling mediated by spinal dynorphin interneurons, but this novel cre driver line may nevertheless be useful to target a broader population of inhibitory spinal dorsal horn neurons.


Introduction
It is widely recognized that discrete subpopulations of inhibitory neurons within the spinal dorsal horn regulate distinct, yet overlapping, somatosensory modalities.Dynorphin neurons, marked by expression of prodynorphin (Pdyn), are key for the suppression of mechanical pain and itch, as their loss or ablation evokes mechanical allodynia and enhanced scratching in response to a range of pruritogens [1][2][3].Importantly, GABAergic synapses originating from spinal dynorphin interneurons provide the majority of inhibitory synaptic input onto lamina I spinoparabrachial projection neurons [4], suggesting that inhibitory dynorphin neurons are key modulators of ascending nociceptive transmission at the level of the spinal cord.
Although dynorphin neurons are easily identified and targeted through the use of an existing Pdyn-IRES-cre driver mouse line [5], the dynorphin population in the superficial dorsal horn (SDH) is not solely composed of inhibitory neurons.Excitatory neurons comprise approximately 30% of dynorphin neurons in laminae I-II of the SDH, with additional glutamatergic dynorphin neurons found in lamina III [6,7], which prevents the straightforward use of this driver line to selectively dissect the role of the inhibitory subset of this mixed population.Indeed, it is likely that the itch-suppressing function of spinal dynorphin interneurons is achieved primarily through GABAergic neurons, while mechanical hypersensitivity may be modulated at least in part by excitatory dynorphin neurons [7].Moreover, dynorphin-expressing neurons are also found in the dorsal root ganglia (DRG; [8,9]) and several brain regions [10][11][12], which necessitates the use of dual-recombinase-dependent intersectional genetic approaches [3,4] or spatially restricted administration of genetic payloads (e.g., through intraspinal injection of viral vectors; [13,14]) to selectively manipulate gene expression in inhibitory dynorphin neurons solely at the level of the spinal cord.
Gucy2d, encoding the membrane-bound guanylate cyclase D (GC-D), has recently emerged as a highly enriched and highly specific marker of inhibitory dynorphin neurons in the spinal dorsal horn, with no Gucy2d mRNA detected in either the dorsal root ganglia (DRG) or brain [15][16][17].Previously, Gucy2d expression had only been documented in a small population of olfactory sensory neurons in the cul-de-sacs of caudal olfactory turbinates [18][19][20] and in axons originating from these neurons which project to the necklace glomeruli in the caudal olfactory bulb [19,21,22].In the main olfactory epithelium (MOE), GC-D may be involved in carbon dioxide detection [20,23] or socially transmitted food preference [24,25], but its function in the spinal cord is not known.Nevertheless, 94% of Gucy2d-expressing cells in the SDH co-express Pdyn mRNA, and virtually all express the inhibitory marker gene Pax2 [17].Moreover, Gucy2d-expressing cells make up about half of dynorphin-lineage neurons in lamina I-III [15] and thus account for a substantial proportion of inhibitory dynorphin neurons [6].This raises the possibility that Gucy2d may provide an avenue to genetically target the inhibitory subpopulation of spinal dynorphin neurons in a straightforward manner, thereby permitting chemogenetic or optogenetic manipulation of these cells with the goal of elucidating their role in modulating pain and/or itch while eliminating unwanted off-target effects of gene expression in other areas of the central and peripheral nervous systems.
Here, we describe a novel Gucy2d-cre driver mouse line that was designed to permit the selective targeting and manipulation of spinal inhibitory dynorphin neurons.Gucy2d-cre mice created via CRISPR/Cas9 genomic knock-in were bred to mice expressing a cre-dependent reporter (either tdTomato or nuclear Sun1.GFP fusion protein), and the resulting offspring were characterized.We found that this driver line labels a subpopulation of inhibitory neurons largely, but not exclusively, located in laminae I-III of the SDH while sparing the DRG, although the majority of these spinal neurons did not co-express Gucy2d mRNA.Moreover, cre-dependent reporter expression was observed in several brain regions, which dramatically reduces the utility of this line in terms of spatial selectivity for the SDH.Although these results preclude the use of this novel line as tool to selectively manipulate inhibitory dynorphin neurons at the level of the spinal cord, Gucy2d-cre mice may nevertheless prove useful in other applications within the neuroscience field.

Animals
All animal experiments were performed in accordance with Institutional Animal Care and Use Committee policies at the University of Cincinnati and Johns Hopkins University.The protocol was approved by the Institutional Animal Care and Use Committee of the University of Cincinnati (Protocol Number 23-02-24-02).All efforts were made to minimize suffering.
Gucy2d-cre mice were generated at the Johns Hopkins Transgenic Mouse Core using CRISPR/Cas9 genomic editing.Single-cell C57BL/6J embryos were injected with CRISPR reagents including Cas9 mRNA, sgRNA with sequence 5'-GCAGACTCACCTGCCATGATG GG-3', and Gucy2d-cre repair plasmid.The chosen sgRNA induced a double-stranded break at the start codon of the Gucy2d-cre open reading frame (ORF) at the end of Exon 1 of the Gucy2d gene.The Gucy2d-cre repair plasmid encoded cre recombinase and B-globin polyA signal flanked by 200-base pair homology arms to encourage homology-directed repair at the site of the double-stranded break, thereby knocking cre recombinase into the Gucy2d ORF.The injected embryos were implanted into pseudopregnant ICR females, and samples from the resulting offspring (i.e., potential founders) were genotyped using primers F-CTCTCTCGTGTGGATCCCCA and R-TGCATCGACCGGTAATGCAG to confirm correct insertion of cre recombinase.Cre-positive founders with correct insertion were bred with mice homozygous for either cre-dependent tdTomato expression (Ai9 mice; Jackson stock #007909) or cre-dependent Sun1.GFP expression (Jackson stock #021039) to facilitate validation and characterization of Gucy2d-cre expression using either of these fluorescent reporters.Cre-negative Ai9 and Sun1.GFP mice (n = 3 Ai9; n = 3 Sun1.GFP) did not exhibit reporter expression in the lumbar spinal cord or brain, indicating a lack of cre-independent (i.e., "leaky") expression (S1 Fig A -H). Gucy2d-cre +/-;Rosa26-LSL-tdTomato +/-offspring are referred to as Gucy2d-tdTomato mice, and Gucy2d-cre +/-;Rosa26-LSL-Sun1.GFP +/-offspring are referred to as Gucy2d-Sun1.GFP mice.Although four cre-positive potential founder Gucy2dcre mice (2 male and 2 female) and their progeny were analyzed, one single male founder was ultimately selected for further characterization in this study.All data and images in this manuscript are derived from that single founder and its progeny.

Tissue preparation, immunohistochemistry, and in situ hybridization
Adult (8-12 weeks) Gucy2d-tdTomato or Gucy2-Sun1.GFP mice of either sex were euthanized via sodium pentobarbital overdose and transcardially perfused with 0.1M phosphate buffer (PB) followed by 4% paraformaldehyde (PFA) in PB.Dorsal root ganglia (DRG) were postfixed for an additional hour in 4% PFA, lumbar spinal cords were postfixed for an additional 2 hours, and brains were postfixed for an additional 6 hours.For main olfactory epithelium, noses were stripped of extraneous skin and muscle tissue, then decalcified in 0.5M EDTA for 3 nights at 4˚C.Fixed tissue was transferred to 30% sucrose in RNAse-free 0.01M phosphatebuffered saline (PBS) and stored overnight at 4˚C.Tissue sections were cut on a Leica 1860CM cryostat and mounted on SuperFrost Plus slides (Fisher).Gucy2d-tdTomato mice were used for anatomical characterization of sites of Gucy2d-cre expression, while Gucy2d-Sun1.GFP mice were used for in situ hybridization and immunohistochemistry experiments owing to easier visualization of the nuclei of cre-expressing cells.

Image acquisition and analysis
Images were captured on a BZ-X810 inverted fluorescent microscope (Keyence) or a BX63 upright fluorescent microscope (Olympus) using CellSens Dimension Software (Olympus).Images obtained under 20X or 40X magnification were acquired as Z-stack images and projected as Extended Focal Images/Full-Focus Images.Lower magnification images were acquired using 4X or 10X magnification and a single focal plane.For quantitative in situ and immunohistochemistry experiments, 3-4 non-adjacent lumbar spinal cord sections from each of 4 mice were evaluated.Cells were considered to be positive for a given in situ hybridization target only if 3 or more puncta in the appropriate fluorescent channel were detected within or touching the boundary of the GFP-labeled nucleus of that cell.DAPI was used as an additional nuclear stain.Full quantification data and images are deposited in the Open Science Framework repository at DOI 10.17605/OSF.IO/G6BV9.

Results
Gucy2d-cre mice were generated by using CRISPR/Cas9 genomic editing to insert DNA encoding cre recombinase at the start codon of the Gucy2d open reading frame located at the end of Exon 1 (Fig 1A).C57BL/6J zygotes were injected with CRISPR components including mRNA encoding Cas9, guide RNA (sgRNA), and Gucy2d-cre repair plasmid containing sequences for cre recombinase and B-globin polyA signal flanked by 200-bp homology arms to facilitate homology-directed repair of the induced double-stranded break.The zygotes were then implanted into pseudopregnant dams.Since the pronuclear injection of CRISPR components was performed at the single-cell stage, the resulting offspring were not chimeric and were therefore genotyped for the presence of the Gucy2d-cre insertion sequence.On this basis, two male and two female potential founders carrying the Gucy2d-cre allele were bred to Ai9 reporter mice, which express tdTomato in a cre-dependent manner.Three out of the four potential founder mice produced Gucy2d-tdTomato offspring with no obvious differences in cre-dependent reporter expression.The fourth founder, a male, produced offspring which were highly variable in terms of cre-dependent reporter expression, with some exhibiting expression that was largely similar to the offspring of the other founders while their littermates exhibited little to no tdTomato expression despite carrying the Gucy2d-cre mutant allele.Due to this inconsistency, this founder and its offspring were excluded from the study.In all other Gucy2d-tdTomato mice examined, the pattern of expression was highly consistent and showed no obvious differences between male and female littermates nor any differences based on whether the sex of the cre-donating parent was male or female.A single founder male was selected to establish the line, and all data shown and described in this manuscript are derived from the progeny of that founder.
The main olfactory epithelium (MOE), spinal cord, brain, and dorsal root ganglia (DRG) of the resulting Gucy2d-tdTomato mice were assessed by histology to determine whether cre-dependent tdTomato expression was consistent with previous characterizations of Gucy2d mRNA or its encoded protein guanylate cyclase D (GC-D) expression in these tissues [15,[17][18][19].As expected, Gucy2d-tdTomato mice exhibit cre-dependent reporter expression in a small number of neurons in MOE turbinates (Fig 1B ), and tdTomato-labeled glomeruli were also observed in the caudal olfactory bulb, consistent with the location of necklace glomeruli (Fig 1C -1D).
A population of cells in the spinal superficial dorsal horn (SDH) of Gucy2d-tdTomato mice was labeled with cre-dependent reporter expression at the cervical, thoracic, and lumbar levels (Fig 2A -2C), which was consistent with previous studies in which Gucy2d mRNA was detected in a subset of spinal dynorphin-lineage SDH neurons [15,17].Also consistent with previous studies, no cre-dependent reporter expression was observed in the DRG (Fig 2D).
To characterize the SDH cells labeled by Gucy2d-cre dependent reporter expression, Gucy2d-cre mice were bred to mice expressing a Sun1-GFP fusion protein in a cre-dependent manner, such that the nuclei of cre-expressing cells (or cells that had previously expressed cre at one point in development) were labeled with GFP.Our earlier examinations of cre-dependent reporter expression in Gucy2d-tdTomato mice, in which the morphology of cells could be more clearly visualized, indicated that the population of spinal cord cells labeled by the Gucy2d-cre mouse line was likely neuronal; however, we confirmed this by performing immunohistochemistry for the neuronal marker NeuN.Indeed, NeuN staining was present in 93.03% ± 1.05% of Gucy2d-Sun1.GFP nuclei (Fig 3A and 3D).Next, to determine whether the labeled neurons were excitatory or inhibitory, we performed in situ hybridization for Slc17a6, which encodes the vesicular glutamate transporter VGLUT2 and is expressed by virtually all excitatory neurons in the SDH [29,30].Gucy2d-Sun1.GFP cells were overwhelmingly inhibitory, with only 7.49% ± 0.99% co-expressing Slc17a6 (Fig 3B and 3D).Finally, we performed in situ hybridization experiments to quantify the colocalization of Gucy2d and/or Pdyn (encoding preprodynorphin) mRNA within the Gucy2d-Sun1.GFP population in the dorsal horn.Gucy2d mRNA was strongly expressed in a subpopulation of cells located in lamina I and II of the SDH, with a few scattered cells located in lamina III.75.08% ± 1.90% of cells expressing Gucy2d mRNA exhibited Sun1.GFP+ nuclei, indicating that most of the Gucy2dexpressing population is captured by this mouse line (Fig 3C and 3D).However, while Gucy2d mRNA was mainly localized to lamina I and the superficial portion of lamina II, a substantial number of Gucy2d-Sun1.GFP nuclei were clearly present in lamina III and deeper dorsal horn laminae, where no Gucy2d mRNA was detected.Accordingly, Gucy2d mRNA was detected in only a minority of the SDH cells labeled by this mouse line (38.27%± 2.28%; As is common with breeding strategies involving a cre driver mouse line paired with a credependent reporter mouse, we hypothesized that this seemingly extraneous population of cells labeled by the Gucy2d-cre mouse line was due to transient expression of Gucy2d (and therefore cre recombinase) during development, leading to reporter expression which persisted even after Gucy2d was no longer actively transcribed.Thus, the cells labeled by cre-dependent reporter in Gucy2d-tdTomato (Fig 4A ) and Gucy2d-Sun1.GFP mice compose the full Gucy2d lineage, rather than reflecting active Gucy2d expression.To test this hypothesis, we injected an AAV8-CAG-FLEX-tdTomato viral vector expressing tdTomato in a cre-dependent manner into the dorsal spinal cords of adult Gucy2d-cre mice.Three weeks later, tdTomato expression was present in a pattern similar to that which was achieved when breeding Gucy2d-cre mice to a cre-dependent reporter mouse line, with reporter expression clearly present in lamina III and in deeper laminae of the spinal dorsal horn (Fig 4B).This suggests that this deeper population of cells is likely still expressing Gucy2d-cre in adulthood (albeit at levels too low to detect with in situ hybridization) which is sufficient to induce recombination and thereby permit virally driven cre-dependent reporter expression.Cre-negative mice injected with the same viral vector did not exhibit tdTomato expression in the spinal cord (S2 Fig A ). Finally, brains of Gucy2d-tdTomato mice were cryosectioned and examined to verify a lack of cre-dependent reporter expression that was expected based on previous mRNA studies [17].We observed tdTomato-labeled cells in the spinal trigeminal nucleus (Sp5) and, surprisingly, in other brain sites including the cerebellum, thalamus, somatosensory cortex, and anterior cingulate cortex (ACC; Fig 5A -5D).The ACC only exhibited a small number of tdTomatoexpressing cells, but expression in the cerebellum, thalamus and somatosensory cortex was quite widespread.To determine whether the cre-dependent reporter expression seen in the brain was the result of transient Gucy2d expression earlier in life, adult Gucy2d-cre mice were intravenously administered the cre-dependent viral vector AAV-PHP.eB-CAG-FLEX-tdTomato.Three weeks later, cre-dependent tdTomato expression was observed in the same brain regions identified in Gucy2d-tdTomato mice (Fig 5E -5H).These results suggest that, similar to our findings in the spinal cord, the presence of cells labeled by Gucy2d-cre despite lacking detectable Gucy2d mRNA is not explained by the transient developmental expression of Gucy2d, but rather to low levels of active expression in adulthood.Cre-negative mice intravenously administered the same viral vector did not exhibit tdTomato expression in any of these brain regions (S2 Fig B -F).

Discussion
Inhibitory spinal cord neurons expressing the opioid peptide dynorphin are key inhibitory modulators of lamina I spinoparabrachial projection neurons [4,31].As such, they are well positioned to dampen ascending nociceptive transmission, and have indeed been shown to play a role in the suppression of mechanical pain and itch [1][2][3][4].However, dynorphin neurons in the spinal dorsal horn (SDH) are not a homogeneous population and may in fact be divided into multiple subpopulations with distinct molecular phenotypes [16].At the most fundamental level, spinal dynorphin neurons are not even uniformly inhibitory, with a substantial glutamatergic subpopulation composing up to 30% of the total dynorphin population in the lumbar spinal cord [6].This poses experimental challenges to targeting inhibitory dynorphin neurons for the purpose of investigating their contributions to nociceptive or pruriceptive processing.
Despite the remarkable selectivity of Gucy2d mRNA expression for spinal dynorphin neurons, Gucy2d knockout mice do not exhibit altered behavioral responses to nociceptive or pruriceptive stimuli under naïve conditions [17].Moreover, putative GC-D receptor ligands guanylin, uroguanylin, CO 2 and CS 2 have been explored in the context of olfaction and socially transmitted food preference [20,23,24,32], but which of these ligands-if any-are operant at the level of the spinal cord is not known.However, we hypothesized that the population of cells which express this gene could nevertheless be involved in modulating the activity of ascending lamina I spinoparabrachial neurons, and thereby pain and itch, through traditional GABAergic inhibitory mechanisms that do not depend on the activities of the GC-D receptor itself.
Multiple genetic strategies have previously been employed to target inhibitory dynorphin neurons in the spinal dorsal horn, which yielded distinct yet overlapping effects on somatosensation.For example, the genetic intersection of the Lbx1 and Pdyn populations selectively targets inhibitory dynorphin neurons throughout lamina I-III of the spinal cord while sparing the DRG and most brain regions, and the ablation of this population induced static and dynamic mechanical allodynia but not itch [3].However, as ablation was carried out in adulthood, this approach likely also ablates neurons which transiently expressed Pdyn earlier in development, thus complicating the assignation of the observed phenotype specifically to adult dynorphin-expressing neurons.Meanwhile, a genetic model which prevents the development of inhibitory spinal dynorphin neurons by suppression of the transcription factor Bhlhb5 induces spontaneous itch as well as enhanced mechanical sensitivity [1].It is notable that the spinal neurons which fail to develop in the Bhlhb5 knockout model, termed "B5-I neurons," are located mainly in laminae I-II.The dorsoventral location of the Pdyn neuronal population targeted by a given genetic strategy likely influences the somatosensory phenotypes which are observed following the removal of the Pdyn neurons from the SDH network, as prior reports show that Pdyn neurons located deeper in the dorsal horn (i.e.along the lamina II-III border) receive a different pattern of primary afferent input compared to Pdyn neurons residing in laminae I-II [3].The selective localization of Gucy2d mRNA to inhibitory dynorphin-expressing neurons located overwhelmingly in laminae I-II [15] raised the possibility that a Gucy2dcre driver mouse could be used to more precisely interrogate the role of superficial dynorphin neurons in somatosensation by providing a means to reversibly manipulate this population using optogenetics or chemogenetics.
As a result, a Gucy2d-cre mouse was developed to facilitate the straightforward genetic manipulation of spinal inhibitory dynorphin neurons.Cre-dependent reporter expression in the Gucy2d-tdTomato offspring of these mice coincided with the previously documented location and distribution of GC-D+ olfactory sensory neurons [18,19,21,22], as tdTomato+ cell bodies were observed in the MOE along with tdTomato+ axonal glomeruli in the caudal olfactory bulb (Fig 1).Similarly, the lack of cre-dependent reporter expression in the DRG of Gucy2d-tdTomato mice was also consistent with the lack of Gucy2d mRNA expression as shown by in situ hybridization experiments [17].Initial evaluation of spinal cord cre-dependent reporter expression also seemed promising, as Gucy2d-tdTomato cells were observed mainly in the SDH (Fig 2 ), corresponding to the location of most spinal dynorphin neurons and previously documented Gucy2d mRNA expression [6,15].
Unfortunately, further characterization of the cells labeled by cre-dependent reporter expression in the spinal cords of Gucy2d-Sun1.GFP mice revealed that, in addition to ~75% of Gucy2d-expressing cells in the SDH, this driver line also induces cre-dependent recombination in a substantial population of cells in laminae I-III in which no Gucy2d mRNA was detected.Pdyn mRNA was also absent from these Gucy2d-negative/Sun1.GFP-positive cells, leading to the conclusion that the majority of spinal cells labeled by the Gucy2d-cre driver mouse are in fact outside of both the dynorphin and Gucy2d-expressing populations (Fig 3).The precise molecular identity of these labeled cells lacking Gucy2d and Pdyn mRNA is unknown, although the wide majority are inhibitory neurons.To further characterize this population, GFP+ nuclei could be selectively isolated from the lumbar spinal cords of Gucy2d-cre-Sun1.GFP mice using the INTACT method [15,33,34] and sequenced using single-nucleus RNAsequencing [16].On the basis of this study, we would expect this approach to reveal Pdyn-positive and Pdyn-negative subpopulations within the Gucy2d-cre lineage and elucidate the relationship between the Gucy2d-cre population and other inhibitory neuronal populations previously identified in the lumbar spinal cord.
The unexpectedly widespread Gucy2d-cre expression observed in the SDH contraindicate the use of this driver line in experimental approaches utilizing a simple strategy of breeding the cre driver line to a cre-dependent gene of interest for selective expression in spinal inhibitory dynorphin neurons.Moreover, reporter expression was observed in brain regions including the spinal trigeminal nucleus, cerebellum, thalamus, somatosensory cortex, and anterior cingulate cortex.Experiments utilizing injection of a cre-dependent AAV viral vector produced a similar pattern of reporter expression, indicating that the unanticipated labeling of cell populations in the deep dorsal horn and the brain is not due to transient expression of Gucy2d-cre at some early point in development (Figs 4 and 5).
Since this mouse line was developed to facilitate the manipulation of inhibitory spinal dynorphin neurons while sparing the DRG and brain, the supraspinal expression is problematic.The thalamus is prominently involved in somatosensory processing, including the transmission of nociceptive signals to other areas in the brain which ultimately integrate such information into the perception of pain [35,36].Moreover, several cortical areas also contained cells labeled by Gucy2d-cre, including the primary somatosensory cortex (S1) and anterior cingulate cortex (ACC).These areas not only receive input from the thalamus [37], but also play distinct roles in modulating the sensory-discriminative aspect or affective valence of pain, respectively [38][39][40].The Gucy2d-cre expression found in these brain regions adds another layer of potential off-target effects on somatosensation.Experiments using this Gucy2d-cre driver mouse to investigate the contributions of spinal inhibitory dynorphin neurons to pain or itch behaviors would therefore be confounded by unwanted cre-dependent expression in key areas relevant to somatosensory processing in addition to the spinal cord neurons which are the intended targets of manipulation.
The Gucy2d-cre driver line created and described in this study failed to provide a straightforward means to target and manipulate spinal inhibitory dynorphin interneurons, but it may nevertheless be useful for other lines of investigation which seek to target a wider subset of inhibitory spinal cord neurons.Selectivity for the spinal cord may be enhanced through the use of a Flp recombinase driver mouse line in conjunction with dual-recombinase-dependent gene expression.Additionally, the observed pattern of cre-dependent reporter expression in the MOE and olfactory glomeruli suggest that this driver line may have utility for future investigations into how the unique population of olfactory sensory neurons which express GC-D regulate chemosensory processing in vivo.

Fig 1 .
Fig 1.Generation of Gucy2d-cre mice via CRISPR knock-in.(A) sgRNA was designed to target the start codon (blue text) of the Gucy2d open reading frame (ORF), which starts at nucleotide 95 of Exon 1.A DNA sequence encoding Cre recombinase flanked by 200-bp homology arms was inserted at the double-stranded break (dashed line) through homology-directed repair.Thus, Cre recombinase is knocked into Exon 1 of the Gucy2d gene.PAM, protospaceradjacent motif.(B) Gucy2d-tdTomato mice exhibit cre-dependent reporter expression in olfactory neurons located in the cul-de-sac regions of the main olfactory epithelium (MOE), as would be predicted based on prior studies of Gucy2d expression.Scale bar = 50 μm.(C, D) Similarly, tdTomato+ axons originating from MOE neurons terminate in the necklace glomeruli in the caudal olfactory bulb.Scale bar in panel C = 100 μm; D = 50 μm.https://doi.org/10.1371/journal.pone.0300282.g001

Fig 2 .
Fig 2. Gucy2d-cre driven reporter expression labels a subset of cells in the spinal dorsal horn while sparing the dorsal root ganglia.Gucy2d-tdTomato mice exhibit cre-dependent reporter expression in the superficial dorsal horn of the cervical (A), thoracic (B) and lumbar (C) spinal cord, which is most prevalent in laminae I-III.Scale bar in panels A-C = 500 μm.(D) Cre-dependent reporter expression is absent from the dorsal root ganglia (DRG) of Gucy2d-tdTomato mice.Scale bar = 100 μm.https://doi.org/10.1371/journal.pone.0300282.g002

Fig 4 .
Fig 4. Cre-dependent reporter labeling in the deeper dorsal horn is not due to transient cre expression during development.(A, B) The pattern of tdTomato expression driven by Gucy2d-cre when crossed to the Ai9 reporter mouse line (A) was similar to tdTomato expression driven by Gucy2d-cre after intraspinal injections of AAV8-CAG-FLEX-tdTomato viral vector in adulthood (B).In both cases, tdTomato+ neurons are visible in laminae III and in deeper dorsal horn laminae.Scale bars in panels A-B = 200 μm.https://doi.org/10.1371/journal.pone.0300282.g004